Acceleration and increased severity of collagen-induced arthritis in P-selectin mutant mice.

P-selectin plays an important role in leukocyte adherence to microvascular endothelium and is expressed in synovial tissue from patients with rheumatoid arthritis (RA). However, the contribution of P-selectin to the initiation and chronicity of joint inflammation is not well understood. In these studies, collagen-induced arthritis (CIA) was induced in P-selectin mutant (-/-) mice to explore the role of P-selectin in the development of joint inflammation.

Anti-inflammatory/antiarthritic ketonic bisphosphonic acid esters.

Bisphosphonate ester 2 is an inhibitor of inflammation, but is devoid of antiarthritic effects. SAR studies on a series of related bisphosphonate esters resulted in compounds 6e, 6i, 6j, and 6m, which exhibited excellent inhibition of an arthritis model, in addition to potent anti-inflammatory effects.

Involvement of intercellular adhesion molecule-1 in the antigen-induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation.

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice.

Future therapies for rheumatoid arthritis: remedies from the horns of a dilemma?

Drugs used to treat arthritis can be broadly classified into either anti-inflammatory or immunosuppressive/immunomodulatory agents. Non-steroidal anti-inflammatory drugs (NSAIDs) are used for both osteoarthritis (OA) and rheumatoid arthritis (RA), while drugs with an immunological mechanism of action are only applicable to RA. The total market value for anti-arthritic drugs is estimated to be 8.

Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhesion molecule-1.

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation.

Inhibition of delayed-type contact hypersensitivity in mice deficient in both E-selectin and P-selectin.

Leukocyte rolling and emigration in response to inflammatory stimuli appears to involve both E-selectin- and P-selectin-dependent adhesion, which suggests that these molecules have overlapping functions. To clarify their relative contributions in chronic inflammation, we examined delayed-type contact hypersensitivity (DTH) responses in P-selectin, E-selectin, and E-/P-selectin-deficient mice. Oxazolone-induced increases in ear thickness and ear weight were equivalent in wild-type mice and in P-selectin and E-selectin mutants, but were significantly reduced in E-/P-selectin mutants.

Reduced susceptibility to collagen-induced arthritis in mice deficient in intercellular adhesion molecule-1.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the firm adhesion of leukocytes to venular endothelium and facilitates leukocyte extravasation from the vasculature into inflamed tissue. In addition, ICAM-1 is an important costimulatory molecule during Ag presentation to lymphocytes. Using mice deficient in ICAM-1, we have investigated the role of this molecule in the development of collagen-induced arthritis.

Role of intercellular adhesion molecule-1 in antigen-induced lung inflammation in brown Norway rats.

We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1; CD54) in ovalbumin (OA) antigen-induced lung inflammation in sensitized Brown Norway (BN) rats by using flow cytometry and in vivo treatment with a murine monoclonal antibody (MAb), 1A29, directed against rat ICAM-1. OA-challenge induced an eosinophil and lymphocyte-rich accumulation of leukocytes into the airway lumen. Between 75 and 90% of the T cells in bronchoalveolar lavage (BAL) fluid after challenge expressed CD54 and CD11a and were of the memory phenotype.

Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized brown Norway rats.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells.

Carbonyl-containing bisphosphonate esters as novel antiinflammatory and antiarthritic agents.

A study of the decomposition of the pyrazoline bisphosphonate ester 2 identified 3 as the sole bisphosphonate component. Evaluation in a delayed-type hypersensitivity granuloma model of chronic inflammation in mice (DTH-GRA) showed 3 to be a potent inhibitor of granuloma formation (sc, 10 mg/kg, 45%), but in a murine model of antigen-induced arthritis (AIA), no significant inhibition was observed. As a result, new ketonic bisphosphonate tetraethyl esters were synthesized from vinylidenebisphosphonic acid tetraethyl ester 4 and activated carbonyl compounds in 13-84% yield.

Demonstration of novel anti-arthritic and anti-inflammatory effects of diphosphonates.

Diphosphonates (DP) are synthetic pyrophosphates with a P-C-P backbone and are predominantly used for the treatment of bone diseases. Several DP have also been shown to exert significant antiarthritic effects in the rat adjuvant-induced polyarthritis model; however, there is no direct evidence for the anti-inflammatory effects of these compounds. We therefore tested the effects of dichloromethylene diphosphonate on delayed-type hypersensitivity granuloma elicited by s.

The effect of an interleukin-1 receptor antagonist protein on type II collagen-induced arthritis and antigen-induced arthritis in mice.

Objective: To investigate the anti-arthritic effect of recombinant human interleukin-1 receptor antagonist protein (IRAP) in two experimental models of arthritis.

Methods: Recombinant IRAP was administered daily to mice with type II collagen-induced arthritis (CIA) or with antigen-induced arthritis (AIA) provoked by methylated bovine serum albumin (mBSA). Disease incidence and severity were assessed by a clinical index and histologic features.

Pyrazoline bisphosphonate esters as novel antiinflammatory and antiarthritic agents.

Vinylidenebisphosphonic acid tetraethyl ester (1) and diazo ketones 7a-1 in ether at 22 degrees C yield pyrazoline bisphosphonate tetraethyl esters 8a-1 in moderate to good yield. These compounds were evaluated in animal models of arthritis: rat adjuvant-induced polyarthritis (AIP) and murine antigen-induced arthritis (AIA) and a murine model of chronic inflammation, the delayed type hypersensitivity granuloma reaction (DTH-GRA). (5-Benzoyl-2,4-dihydro-3H-pyrazol-3-ylidene)-bisphosphonic acid tetraethyl ester (8a), and [5-(3-fluorobenzoyl)-2,4-dihydro-3H-pyraxol-3-ylidene]- bisphosphonic acid tetraethyl ester (8d) significantly inhibited the arthritis models, AIP (15 mg/kg) and AIA (25 mg/kg), as well as the DTH-GRA (25 mg/kg).

The effects of several diphosphonates on murine bone marrow proliferation.

Six diphosphonates were examined for their ability to alter proliferative responses of mouse bone marrow cells to recombinant human M-CSF and recombinant murine GM-CSF. Risedronate ([2-(3-pyridinyl)-ethylidene] hydroxy bisphosphonic acid) added to in vitro cultures at 10 microM, suppressed the response to M-CSF by 58%, but had no significant effect on GM-CSF-induced proliferation. Ethane-1-hydroxy-1,1-bisphosphonic acid (EHDP), dichloromethylene bisphosphonic acid (Cl2MBP), 3-amino-1-hydroxy-propylidene-1,1-bisphosphonic acid (APD), (4-chlorophenyl)-thiomethylene bisphosphonic acid (tiludronate) and (cycloheptylamino)-methylene bisphosphonic acid (YM-175) had no significant effect.

Modulation of T lymphocyte function by the angiogenesis inhibitor AGM-1470.

The angiogenesis inhibitor AGM-1470 has recently been reported to inhibit collagen-induced arthritis in rats. To determine if the anti-arthritic effects of AGM-1470 might be due to T cell inhibition, we have studied its effects on T cell responses in vitro. Responses of human cells to tetanus toxoid (TT), and those of murine splenocytes to staphylococcal enterotoxin (SE), mitogens or a mls difference were inhibited by AGM-1470.

Chemical modification of interleukin-1 beta: biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein.

We have modified recombinant human Interleukin-1 beta using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide at pH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI = 6.

Site-specific chemical modification of interleukin-1 beta by acrylodan at cysteine 8 and lysine 103.

Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate the probe on the modified proteins.

Induction of an acute erosive monarticular arthritis in mice by interleukin-1 and methylated bovine serum albumin.

We examined the effect of interleukin-1 (IL-1) administration on a mild and transient inflammatory response in the knees of mice injected intraarticularly with methylated bovine serum albumin (mBSA). Injection of mBSA on day 0 into nonsensitized mice caused a weak inflammatory response confined to the infrapatellar fat pads and involved infiltration by mononuclear cells, neutrophils, and eosinophils. The response developed between days 4 and 7 and resolved by day 28.

Biotinylation of reactive amino groups in native recombinant human interleukin-1 beta.

Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94.

The effects of sensitization protocols on arthritic responses in antigen-induced arthritis.

Three different immunization protocols were studied for their effect on methylated bovine serum albumin (mBSA)-induced arthritis. Mice were immunized with mBSA in Freund's incomplete adjuvant containing dextran and challenged 3 weeks later with antigen in one knee. After 7-14 days a strong inflammatory reaction developed without significant erosions of articular cartilage or bone.

Isolation and bioactivities of three IL-1 beta N-terminal variants.

Three distinct N-terminal variants of rhIL-1 beta can be generated by expression of the IL-1 beta gene in E. coli; the naturally occurring Ala1 species, Met0-Ala1 and des-Ala1 proteins. Since most studies with rhIL-1 beta have used a mixture of two or more variants, we have evaluated their individual bioactivities.

Soybean trypsin inhibitor. An IL-1-like protein?

Soybean trypsin inhibitor (SBTI) shares some structural homology with interleukin-1 (IL-1) and was tested for IL-1 bioactivity. Human T-cells proliferated maximally when stimulated with PMA and SBTI but failed to respond to either stimulus alone. This response was abrogated by neutralizing antibodies to IL-1 beta but not to IL-1 alpha.

Acute and chronic inflammatory responses to local administration of recombinant IL-1 alpha, IL-beta, TNF alpha, IL-2 and Ifn gamma in mice.

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1 alpha, IL-1 beta, TNF alpha, IL-2 and Ifn gamma. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1 alpha greater than rIL-1 beta greater than rTNF alpha greater than rIfn gamma = BSA (control) following a single sc. injection.

Local pathological responses to slow-release recombinant interleukin-1, interleukin-2 and gamma-interferon in the mouse and their relevance to chronic inflammatory disease.

1. The present study describes the pathological responses to local administration of recombinant cytokines in subcutaneously implanted slow-release ethylene vinyl acetate (EVA) co-polymer in mice. 2.